On the mechanism of DNA transfection: efficient gene transfer without viruses.
From an investigation of how transfected DNA navigates from the cell surface to the nucleus, we have developed a transfection method for primary human fibroblasts that approaches the efficiency of viruses. We have visually tracked the subcellular routing of exogenous DNA and find that all cells in an asynchronous population are surprisingly competent in the nuclear uptake of DNA, but two steps practically limit efficient transfection to a minority of cells. First, regardless of the method used to traverse the cell membrane--CaPO4 precipitation, lipofection or electroporation--it appears that nuclear transport of DNA requires routing through endosomes and lysosomes. Apparent abrogation of endosome-lysosome fusion or translocation with microfilament or microtubule toxins, respectively, inhibits the nuclear accumulation of transfected DNA, but interruption of lysosomal function with protease inhibitors promotes it. Second, in normal human fibroblasts, which are refractory to transfection the exogenous DNA is rapidly excluded from the nucleus, but in HeLa cells, which are readily transfected, there is prolonged nuclear stability of the DNA, indicating the failure in HeLa cells of a mechanism for the elimination of foreign DNA. These observations imply strategies for optimizing gene transfer efficiency in virus-independent approaches to gene therapy.
Coonrod
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Markey Molecular Medicine Center, Department of Medicine, University of Washington, Seattle, USA.
Li
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FQ
Horwitz
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D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003572:Cytochalasins / Q000494:pharmacology; D004247:DNA / Q000032:analysis*; D018274:Electroporation; D011992:Endosomes / Q000737:chemistry*; D005347:Fibroblasts; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D006801:Humans; D008081:Liposomes; D008247:Lysosomes / Q000737:chemistry*; D008297:Male; D014162:Transfection / Q000187:drug effects / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
D015853:Cysteine Proteinase Inhibitors; D003572:Cytochalasins; D008081:Liposomes; D004247:DNA; D001616:beta-Galactosidase